There have been many reports on methods of improving taste and/or flavour using enzymes. For example, as a method for enhancing umami a process of increasing free amino acids by the use of a combination of proteases and peptidases has been known. Such a process may be used for not only producing seasoning mix but also improving the quality and taste of meat (Japanese publication of non-examined patent application (JP-Kokai) No. 05-276899). There is also a method which uses an enzyme that specifically functions on proline as described in JP Kokai No. 11-075765 or No. 07-115969. A method of enhancing umami is also known where glutamate is increased by using a γ-GTP (gamma glutamyl transpeptidase) during protease reaction or by converting glutamine to glutamate by a glutaminase. The peptidases of the present invention were also reported to have an umami-enhancing activity because they specifically release glutamate or aspartate (JP Kokai No. 2000-325090).
For example koji molds have been used to produce natural seasonings including soy sauce, miso and other protein hydrolysates. For example soy sauce is produce through two steps, koji-making and fermentation. The raw materials are mainly hydrolized by enzymes produced by koji molds (Aspergillus fungi) during the koji-making step. During the step, it is important to increase the content of free amino acids in the resulting koji (moromi), particularly the content of free glutamate to improve the taste of soy sauce.
In general, amino acids are generated through two steps from raw material proteins. The first step is the release of peptides from the proteins by proteases, and the second step is the generation of amino acids by hydrolysis of the peptides, which is catalyzed by peptidases.
Asano et al. noticed that soybeans hydrolyze the storage proteins in the seeds in a very short period during the germinating process and found a peptidase (a peptidase which efficiently hydrolyzes peptides containing acidic amino acid residues and leucine aminopeptidases) and succeeded in the efficient hydrolysis of soybean proteins (JP Kokai No. 9-294583).
The soybean aminopeptidase is an enzyme which has been proved to be a novel enzyme, which has not been reported previously, as defined by its enzymatic profile. The existence of the enzyme has not been known except in germinating soybeans. The soybean aminopeptidase has the activity of releasing N-terminal acidic amino acids efficiently from peptides having acidic amino acids such as glutamic acid at the N-terminal. It is known that a di-peptide consisting of two glutamic acid residues exists as a hydrolysis-resistant peptide in seasonings including protein hydrolysates, such as soy sauce. Accordingly, it is possible to hydrolyze such hydrolysis-resistant di-peptides by using the action of the soybean aminopeptidase to produce a seasoning liquid which has a high content of free glutamate and improved taste.
Ninomiya et al. succeeded in the mass production of the soybean aminopeptidase by using genetic recombination techniques (JP Kokai No. 2000-325090), but the soybean aminopeptidase GX produced by this method can be hardly used for producing seasoning liquid, because peptidases from soybean are not approved as enzymes for foods. Additionally, there remain several problems in their applicability because the recombinant soybean is poor in the heat-stability and is not suitable for the reaction at 50° C. or more.
For peptidases from koji molds which include many microorganism species for foods, peptidases from Aspergillus oryzae and Aspergillus sojae have been reported (JP Kokai No. 11-346777, DE95-1952648, WO 98/51163, WO 96/28542, WO 96/15504, WO 98/51803, WO 98/14599). Among them, there are many reports on leucine aminopeptidases, but there is no report on a peptidase that has an activity to efficiently release glutamate, such as the soybean aminopeptidase GX. For example, Koibuchi et al. screened a Aspergillus nidulans genomic DNA library using Aspergillus nidulans ESTs having a homology to the soybean aminopeptidase GX and obtained a DNA encoding a novel aminopeptidase of Aspergillus nidulans (WO 02/077223). However, the obtained novel aminopeptidase was the enzyme having leucine aminopeptidase (LAP) activity, which requires cobalt or zinc ion for its activation, although it had a homology of close to 40% to the soybean GX. Thus, an enzyme having the soybean aminopeptidase-like properties has not been obtained from other sources than soybean. Furthermore, it was shown that the existence of the soybean aminopeptidase-like properties could not be determined from the sequence homology alone.
In this connection, an EST database of a koji mold (Aspergillus oryzae RIB40) was opened on Mar. 31, 2003 at the Web site of National Research Institute of Brewing and the search for the sequences has been possible.
On the other hand, methods for improving sweetness and flavour are known where hydrolyzing enzymes or microorganisms containing the enzymes are used, or a combination of the enzymes and other processes are used for improving sweetness. For example, in JP Kokai No. 09-299094 after reacting the enzymes or the microorganisms on carbohydrates, alcohol fermentation was conducted to improve the flavour. In JP Kokai No. 09-299094, the improvement in sweetness was also succeeded by functions of sugar hydrolyzing enzymes together with sugar transfer reactions and condensation reactions. Particularly, in JP Kokai No. 2003-153651 sweetness and umami were increased by reacting enzymes which decompose tannin, polysaccharides and proteins on tea leafages material or dried tea leafages to reduce astringency. However, a method for enhancing sweetness by the action of peptidases alone has not been reported.
As methods for reducing saltiness edge, treatment with various essences (JP Kokai No. 2002-034496) or yeast (JP Kokai No. 11-276113) or the addition of soybean mineral concentrates (JP Kokai No. 05-049439) have been reported. However no successful examples have been known where saltiness edge taste was reduced by a peptidase treatment.
The following processes have been reported as general methods for improving flavour and taste by enzymes. For improving the taste of egg yolk, phospholipases has been used and JP Kokai No. 2002-325559 clearly describes the effects of phospholipase A1. In JP Kokai No. 2002-253171, bitter amino acids were γ-glutamylated by a γ-glutamyl transpeptidase and the reduction of bitterness, the increase of sourness and the improvement of taste were successful. Additionally, in JP Kokai No. 2000-327692 the taste and the solubility of isoflavones were improved by the function of glycosyltransferases. Besides these reports, many methods are known including a method for producing taste-improving food materials by glutamate decarboxylases (JP Kokai No. 2000-166502), a method for providing flavour improving composition by synthesizing adenine using glutaminases (JP Kokai No. 09-313129), a method for improving flavour of foods using beta-primevelosidases (JP Kokai No. 08-140675), a method for improving the flavour of oils and fats using lipases (JP Kokai No. 07-135972) and a method for improving the taste of bread by a combination of lactic acid bacteria, lipases and proteases. However, a method for improving taste and/or flavour by peptidases alone has not been known.